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1.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
2.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070225

RESUMO

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Peptidoglicano/química , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética
3.
Science ; 372(6546): 1057-1062, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083482

RESUMO

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.


Assuntos
Códon , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/virologia , Compostos Macrocíclicos/metabolismo , Polímeros/metabolismo , Biossíntese de Proteínas , Fagos T/crescimento & desenvolvimento , Aminoácidos/metabolismo , Bacteriólise , Uso do Códon , Códon de Terminação , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Deleção de Genes , Código Genético , Genoma Bacteriano , Compostos Macrocíclicos/química , Mutagênese , Fatores de Terminação de Peptídeos/genética , Polímeros/química , RNA Bacteriano/genética , RNA de Transferência/genética , RNA de Transferência de Serina/genética , Ubiquitina/biossíntese , Ubiquitina/genética
4.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064730

RESUMO

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria-photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.


Assuntos
Escherichia coli/metabolismo , Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Fármacos Fotossensibilizantes/metabolismo , Staphylococcus aureus/metabolismo , Tomografia de Coerência Óptica/métodos , Escherichia coli/crescimento & desenvolvimento , Processamento de Sinais Assistido por Computador , Staphylococcus aureus/crescimento & desenvolvimento
5.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066237

RESUMO

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.


Assuntos
Amiloide/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Agregados Proteicos , Conformação Proteica , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Homologia de Sequência
6.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
7.
Nat Commun ; 12(1): 3312, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083526

RESUMO

Self-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we experimentally show that the Min protein dynamics on the membrane crucially depend on the micro chamber height due to bulk concentration gradients orthogonal to the membrane. A theoretical analysis shows that in vitro patterns at low microchamber height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger microchamber height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work reveals the qualitatively different mechanisms of mass transport that govern Min protein-patterns for different bulk heights and thus shows that Min patterns in cells are governed by a different mechanism than those in vitro.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Polaridade Celular , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Técnicas In Vitro , Modelos Biológicos , Dinâmica não Linear
8.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
9.
Nat Commun ; 12(1): 3419, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103495

RESUMO

Bacteria can be harnessed to synthesise high-value chemicals. A promising strategy for increasing productivity uses inducible control systems to switch metabolism from growth to chemical synthesis once a large population of cell factories are generated. However, use of expensive chemical inducers limits scalability of this approach for biotechnological applications. Switching using cheap nutrients is an appealing alternative, but their tightly regulated uptake and consumption again limits scalability. Here, using mathematical models of fatty acid uptake in E. coli as an exemplary case study, we unravel how the cell's native regulation and program of induction can be engineered to minimise inducer usage. We show that integrating positive feedback loops into the circuitry creates an irreversible metabolic switch, which, requiring only temporary induction, drastically reduces inducer usage. Our proposed switch should be widely applicable, irrespective of the product of interest, and brings closer the realization of scalable and sustainable microbial chemical production.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica , Retroalimentação Fisiológica , Homeostase , Ácido Oleico/metabolismo
10.
Nat Commun ; 12(1): 2805, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990606

RESUMO

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Terapia Biológica/métodos , Microbioma Gastrointestinal , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Fenilcetonúrias/terapia , Animais , Células CACO-2 , Simulação por Computador , Escherichia coli/metabolismo , Engenharia Genética , Células HT29 , Humanos , Técnicas In Vitro , Microfluídica , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/microbiologia , Primatas , Biologia Sintética
11.
Nat Commun ; 12(1): 2641, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976175

RESUMO

The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises longstanding questions about the order of events in one of life's most central processes. Here we follow the reaction pathway of a replicative DNA polymerase using time-resolved X-ray crystallography to elucidate the order and transition between intermediates. In contrast to the canonical model, the structural changes observed in the time-lapsed images reveal a catalytic cycle in which translocation precedes catalysis. The translocation step appears to follow a push-pull mechanism where the O-O1 loop of the finger subdomain acts as a pawl to facilitate unidirectional movement along the template with conserved tyrosine residues 714 and 719 functioning as tandem gatekeepers of DNA synthesis. The structures capture the precise order of critical events that may be a general feature of enzymatic catalysis among replicative DNA polymerases.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Genéticos , Conformação de Ácido Nucleico , Fatores de Tempo
12.
Nat Commun ; 12(1): 2775, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986273

RESUMO

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of ß-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.


Assuntos
Antibacterianos/farmacologia , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , beta-Lactamas/farmacologia , Acetilglucosamina/química , Aldeídos/química , Microscopia Crioeletrônica , Ácidos Murâmicos/química , Oligossacarídeos/farmacologia , Peptidoglicano/biossíntese , Conformação Proteica , Domínios Proteicos/fisiologia
13.
Nat Commun ; 12(1): 2571, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958590

RESUMO

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.


Assuntos
Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endonucleases/química , Integrases/química , Piscirickettsiaceae/química , DNA Polimerase Dirigida por RNA/química , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Piscirickettsiaceae/enzimologia , Piscirickettsiaceae/metabolismo , Proteínas Recombinantes
14.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elongação da Transcrição Genética
15.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1298-1311, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973443

RESUMO

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral ß-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.


Assuntos
Rhodospirillaceae , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Especificidade por Substrato
16.
J Hazard Mater ; 412: 125248, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33951868

RESUMO

Cadmium (Cd) is a typical and widely present toxic heavy metals in environments. Biomineralization of Cd ions could alleviate the toxicity and produce valuable products in certain waste streams containing selenite. However, the impact of the intrinsic Cd(II) efflux system on the biotransformation process remains unrevealed. In this work, the significance of the efflux system on Cd biomineralization was evaluated by constructing engineered Escherichia coli strains, including ΔzntA with suppressed Cd(II) efflux system and pYYDT-zntA with strengthened Cd(II) efflux system. Compared to the wild type (WT), 20% more Cd ions were accumulated in ΔzntA and 17% less were observed in pYYDT-zntA in the presence of selenite as determined by inductively coupled plasma atomic emission spectrometer. Through combination with X-ray absorption fine structure analysis, it was discovered that 50% higher production of CdSxSe1-x quantum dots (QDs) was achieved in the ΔzntA cells than that in the WT cells. Moreover, the ΔzntA cells exhibited the same viability as the WT cells and the pYYDT-zntA cells because accumulated Cd ions were transformed into biocompatible QDs. In addition, the biosynthesized QDs had a uniform particle size (3.82 ± 0.53 nm) and a long fluorescence lifetime (45.6 ns), which could potentially be utilized for bio-imaging. These results not only elucidate the significance of Cd(II) efflux system in the biotransformation of Cd ions and selenite, but also provide a promising way to recover Cd and Se as valuable products in certain waste streams.


Assuntos
Proteínas de Transporte de Cátions , Pontos Quânticos , Biomineralização , Cádmio/metabolismo , Cádmio/toxicidade , Cátions , Escherichia coli/genética , Escherichia coli/metabolismo
17.
Nat Commun ; 12(1): 3204, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050162

RESUMO

Despite mounting evidence that in clonal bacterial populations, phenotypic variability originates from stochasticity in gene expression, little is known about noise-shaping evolutionary forces and how expression noise translates to phenotypic differences. Here we developed a high-throughput assay that uses a redox-sensitive dye to couple growth of thousands of bacterial colonies to their respiratory activity and show that in Escherichia coli, noisy regulation of lower glycolysis and citric acid cycle is responsible for large variations in respiratory metabolism. We found that these variations are Pareto optimal to maximization of growth rate and minimization of lag time, two objectives competing between fermentative and respiratory metabolism. Metabolome-based analysis revealed the role of respiratory metabolism in preventing the accumulation of toxic intermediates of branched chain amino acid biosynthesis, thereby supporting early onset of cell growth after carbon starvation. We propose that optimal metabolic tradeoffs play a key role in shaping and preserving phenotypic heterogeneity and adaptation to fluctuating environments.


Assuntos
Adaptação Fisiológica/genética , Processos de Crescimento Celular/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Ciclo do Ácido Cítrico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Glicólise/genética , Processos Estocásticos
18.
Plant Physiol Biochem ; 165: 57-70, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34034161

RESUMO

Universal Stress Protein A (USPA) plays critical roles in the regulation of growth, development and response to abiotic stress in plants. To date, most research related to the role of USPA in plants has been carried out in herbaceous models such as Arabidopsis, rice and soybean. Here, we used bioinformatics approaches to identify 21 USPA genes in the genome of Vitis vinifera L. Phylogenetic analysis revealed that VvUSPAs could be divided into eight clades. Based on predicted chromosomal locations, we identified 16 pairs of syntenic, orthologous genes between A. thaliana and V. vinifera. Further promoter cis-elements analysis, together with identification of potential microRNA (miRNA) binding sites, suggested that at least some of the VvUSPAs participate in response to phytohormones and abiotic stress. To add support for this, we analyzed the developmental and stress-responsive expression patterns of the homologous USPA genes in the drought-resistant wild Vitis yeshanensis accession 'Yanshan-1' and the drought-sensitive Vitis riparia accession 'He'an'. Most of the USPA genes were upregulated in different degrees in the two genotypes after drought stress and exposure to ethephon (ETH), abscisic acid (ABA) and methyl jasmonate (MeJA). Individual USPA genes showed various tissue-specific expression patterns. Heterologous expression of five selected genes (VvUSPA2, VvUSPA3, VvUSPA11, VvUSPA13 and VvUSPA16) in Escherichia coli (E. coli) enhanced resistance to drought stress. Our study provides a model for mapping gene function in response to abiotic stress and identified three candidate genes, VvUSPA3, VvUSPA11 and VvUSPA16, as regulators of drought response in V. vinifera.


Assuntos
Vitis , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteína Estafilocócica A , Estresse Fisiológico/genética , Vitis/genética , Vitis/metabolismo
19.
Appl Microbiol Biotechnol ; 105(11): 4609-4620, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34043081

RESUMO

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by ß-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.


Assuntos
Proteínas de Escherichia coli , Proteínas Periplásmicas de Ligação , Permeabilidade da Membrana Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Periplasma/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946710

RESUMO

Considering the advent of antibiotic resistance, the study of bacterial metabolic behavior stimulated by novel antimicrobial agents becomes a relevant tool to elucidate involved adaptive pathways. Profiling of volatile metabolites was performed to monitor alterations of bacterial metabolism induced by biosynthesized silver nanoparticles (bio-AgNPs). Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and Proteus mirabilis were isolated from pressure ulcers, and their cultures were prepared in the presence/absence of bio-AgNPs at 12.5, 25 and 50 µg mL-1. Headspace solid phase microextraction associated to gas chromatography-mass spectrometry was the employed analytical platform. At the lower concentration level, the agent promoted positive modulation of products of fermentation routes and bioactive volatiles, indicating an attempt of bacteria to adapt to an ongoing suppression of cellular respiration. Augmented response of aldehydes and other possible products of lipid oxidative cleavage was noticed for increasing levels of bio-AgNPs. The greatest concentration of agent caused a reduction of 44 to 80% in the variety of compounds found in the control samples. Pathway analysis indicated overall inhibition of amino acids and fatty acids routes. The present assessment may provide a deeper understanding of molecular mechanisms of bio-AgNPs and how the metabolic response of bacteria is untangled.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Nanopartículas Metálicas/uso terapêutico , Lesão por Pressão/tratamento farmacológico , Lesão por Pressão/microbiologia , Prata/uso terapêutico , Compostos Orgânicos Voláteis/metabolismo , Antibacterianos/uso terapêutico , Bactérias/isolamento & purificação , Farmacorresistência Bacteriana , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Metabolômica , Testes de Sensibilidade Microbiana , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/isolamento & purificação , Proteus mirabilis/metabolismo , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/classificação
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